Ion channel measurement plays an important role in determining the effect of compounds and the like at the cellular level. Ion channels are transmembrane proteins that form pores in a cell membrane, which, in turn, allow ions to pass from one side of the cell membrane to another. The activity of these channels influences cell membrane potential and are responsible for mediating changes in overall cell excitability.
Patch clamp techniques are commonly used for performing ion channel measurements. Certain patch clamp techniques are described in Sakmann and Neher, “Single-Channel Recording,” Plenum, 1995. Typically, such techniques involve the use of a glass pipette or micropipette to contact with, and ideally attach to, a cell. A conventional micropipette for patch clamp recording is a hollow glass tube having a polished blunt tip and an aperture therethrough. Other micropipette types, such as sharp electrodes, are employed in other types of electrophysiology. Sharp microelectrodes typically impale cells rather than forming tight seals with the membrane. Methodologies for forming micropipettes are disclosed in Brown and Flaming, “Advanced Micropipette Techniques for Cell Physiology”, 1989, and a cross-section of a blunt tip of a prior art micropipette is displayed in FIG. 1.
To have the micropipette contact with, and attach to, the cell, standard cell location and pipette placement techniques may be used. Alternatively, the cell may be positioned, for example, at the aperture of the micropipette by manipulating an electric field via an electrode positioned in proximity to the aperture. A critical step in any patch clamp recording process is the formation of an acceptable seal between the outside surface or inner rim of the blunt tip of the micropipette and the attached cell. An acceptable seal is often referred to as a “giga-ohm seal” because it generally demonstrates electrical resistance measurable in giga-ohms. (Reference herein to forming a “seal” shall mean the formation of an electrically acceptable seal having sufficient electrical resistance to be measurable; it is preferred that the electrical resistance be greater than or equal to 1 giga-ohm, but may be less than.) Conventional techniques are known for forming a seal.
After seal formation, it is often desired to obtain “whole cell” access which is achieved via electroporation, additional negative pressure, chemopermeabilization or a combination thereof. (Mere attachment of the micropipette, even in a sealed state, to the cell provides a limited “on cell” configuration which can be used to measure ion channel properties from the small patch of membrane under the electrode). Test compounds and the like may then be applied to the cell, and changes in its electrical properties (i.e., current, potential, conductance and capacitance) may thereafter be measured. These measurements, in turn, can be used to assay ion channel activity, transporter activity, or changes in the properties of cell membrane caused by cell-to-cell interactions, cell-to-cell fusion, viral infection, endocytosis, exocytosis, membrane cycling and membrane-to-ligand interactions.
Various patch clamp techniques employing blunt tip micropipettes are disclosed in U.S. Pat. No. 6,063,260 and PCT Publication Nos. WO 98/50791, WO 99/66329 and WO 00/34776.
FIG. 1 displays an entire blunt tip pipette 10 and an enlarged cross-section of the tip thereof, as found in conventional technologies. The pipette 10 typically has an aperture 12 disposed at the tip 11 thereof. Such pipettes 10 are typically composed of a glass. To perform patch clamping, the aperture 12 is positioned to contact a subject cell. In the prior art, the act of positioning the micropipette tip is tedious, requiring initial cell location (e.g., on a dish, in a cell bath); and, positioning the micropipette properly relative to the located cell. With prior art techniques, the processes of cell location and micropipette positioning are manually achieved, typically using a high quality microscope, and a high quality three axis micromanipulator (with sub-micro resolution in each axis and a high degree of stability) to control location of the micropipette tip. As is readily appreciated, the skill of the operator determines the speed and efficiency by which patch clamping can be conducted. Techniques have been developed to facilitate the cell contacting, such as with introducing a slight electrical field at the tip of the micropipette. In addition, techniques have been developed which, through automation, allow for repeated cell location and proper micropipette positioning, such as a technique disclosed in WIPO International Publication Number WO 00/34776; and a technique disclosed in copending U.S. patent application Ser. No. 60/338,715 the inventors and assignee herein. Upon cell location and contacting, seal formation is achieved using known techniques, and electrophysiological properties of the cell are measured by an electrode or other probe, and associated electronics.
Conventional pipette designs, however, suffer from various drawbacks, including the time required for manual fabrication. Since pipettes cannot be reused, time-consuming fabrication is required individually for each cell to be tested. In addition, conventional pipette designs suffer from propendency to become blocked, difficulty in filling with saline solutions (especially close to the tip) and the limitation that seal formation must occur outside the pipette. Similarly electrical measurement can be distorted by the unfavorable electrical properties arising from the narrow opening of blunt tip pipettes. In addition, the processes of cell contacting, attachment, seal formation and electrical measurement require advanced and detailed steps that are not readily amenable to automation. Thus, a high degree of skill is required to perform patch clamp techniques using existing technologies. Accordingly, there is a need for new pipette configurations which readily addresses certain problems of the prior art.